Recent key studies have shown that the composition of the extracellular matrix (ECM) can determine metastatic outcome, specifically whether a tumor cell will transition from a non-invasive to an invasive phenotype. However, the study of ECM-cell interactions has been limited to reductionist methods that focus on one or a few ECM proteins. To date, the extracellular biomolecules that are responsible for influencing this change in cell phenotype are largely unknown. A better understanding of the ECM role in promoting tumor cell metastasis will be facilitated by a more global characterization of ECM composition. Proteomic approaches to study ECM have been hindered by the proteolytic and solubilization resistant properties of highly cross-linked matrix components, making study by more traditional proteomic techniques difficult, if not impossible. A major barrier to progress in this field is the lack of suitable sample preparation methods for effective molecular characterization of ECM. The focus of this grant is the optimization of ECM sample preparation methods. To develop effective sample preparation methods we will establish a reproducible source of ECM. A three- dimensional cell culture model will be used to evaluate the tumor promotional attributes of ECM secreted by two pairs of isogenic human mammary epithelial cell lines. The first areas of sample preparation development will be in the optimization of cell removal from its underlying ECM, with emphasis on eliminating contaminate intracellular proteins that co-purify with the ECM. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) profiling and Western blots will be used to evaluate the level of cellular contaminants. Next, ECM solubilization strategies and effective cleavage methods will be explored using as endpoints the number of distinct ECM protein identified and percent sequence coverage. Utilization of a label-free mass spectrometry based quantification method will provide relative ranking of the strategies and assist in further method optimization. We hypothesize that protein-protein crosslinks influence the effectiveness of matrix sample preparation methods for ECM. The two major classes of proteins that catalyze the cross-linking of ECM proteins show aberrant expression and activity in neoplastic cell lines and various cancerous tissues. We will use recombinant human tissue transglutaminase and its inhibitors to determine how crosslinking influences cell removal, solubilization and digestion efficiencies with emphasis on protein quantification. Cancer cells deposit ECM proteins into their microenvironment that can program non-metastatic cells to a phenotype consistent with metastasis. The development of the proposed cell culture model and state-of the- art ECM specific proteomic techniques will advance our ability to explore and characterize the role of the extracellular matrix in metastasis. These studies will be the foundation for translational experiments, clinical investigations and should ultimately lead to biomarkers and therapeutic approaches that will improve patient care and survivability. PUBLIC HEALTH RELEVANCE: Despite the fundamental role that the cell microenvironment plays in tumor progression, methods to study the underlying molecular mechanisms are lacking. This work is aimed at developing the sample preparation methods necessary to characterize the matrix component of the cell microenvironment so that we may ultimately develop new therapeutic strategies and identify early diagnostic markers of cancer.